Field of the Invention
The present invention relates to the field of biotechnology engineering, and more particularly relates to a method of constructing a recombinant Bacillus subtilis that can produce specific-molecular-weight hyaluronic acids.
Description of the Related Art
Hyaluronic Acid (HA or hyaluronan), a highly viscous polysaccharide, was first isolated from bovine vitreous and it is the best moisturizing substances found in nature. The unique rheological, the viscoelastic and hygroscopic properties along with the biocompatibility and non-immunogenicity has enabled HA to be widely used in cosmetics, food and pharmaceutics. Recent studies have found that HAs with low molecular weight (less than 1×104 Da) and HA oligosaccharides have unique biological functions, for example, HA oligosaccharides with molecular weight less than 1×104 Da are involved in wound healing and tumor cell apoptosis. In addition, the HA oligosaccharides can be easily absorbed by human body and act as precursors for the body's own synthesis of polysaccharides. Therefore, HA oligosaccharides have important applications in the areas of food, health care and medicine.
HA is widely distributed in animal tissues, such as comb, synovial fluid, cartilage and vitreous. And it is also distributed in bacteria, such as Bacillus aerogenes, Pseudomonas aeruginosa and Hemolytic streptococcus. Due to disadvantages (such as the risk of cross-species viral infection) of the traditional HA extraction methods from animal tissues, the commercial HA production is mainly relied on fermentation of certain attenuated strains of group C Streptococcus (such as S. equi and S. zooepidemicus). The HA produced by microbial fermentation was mostly high molecular weight HA. With increasing health and safety requirements, it becomes more and more urgent to find a safe and reliable microbial host for HA production.
Although de novo synthesis methods for preparing HA oligosaccharides have been reported, it is difficult to achieve large-scale production due to high cost of substrates, complex synthesis steps and low yields. Currently, physical and chemical degradation are the main methods used for low-molecular weight HA production, which have many disadvantages, such as generation of HA with a wide range of molecular weight distribution, poor product stability, high cost of purification, high energy consumption and high pollution. Compared to physical or chemical degradation, enzymatic catalytic synthesis of HA oligosaccharides is a promising approach that offers great industrial potential. However, the enzymatic method requires preparation of large amount of hyaluronidase (HAase) and precise control of the enzymatic catalytic reaction conditions. Therefore, constructing a single microbial strain that simultaneously produces both HA and HAase can offer great benefits in HA research and industrial applications.
HAase is a class of hyaluronic acid-degrading enzymes widely distributed in nature. According to sources, structures and function mechanisms of the enzyme, HAase are divided into three categories: endo-β-N-acetyl-glucosaminidase (EC 3.2.1.35, mainly exist in mammals and venom of bees, snakes and spiders), endo-β-glucuronidase enzyme (EC 3.2.1.36, mainly in the leech) and hyaluronic acid lyase (EC 4.2.2.1, mainly exist in bacteria, bacteriophages and fungi). As a “scatter factor”, HAase is widely used as auxiliaries for drug diffusion in clinical applications. However, the commercial HAase obtained from bovine testicular tissue is usually of poor quality and expensive to produce, and has the risk of Animal foci infection.
In the present invention, a HA biosynthetic pathway was constructed in Bacillus subtilis (B. subtilis) and high yield of HA was achieved by regulating the expression of important genes for synthesis of HA precursors, UDP-GlcNAc and UDP-GlcA, in the engineered strain. In addition, Leech HAase was co-expressed in the engineered strain with a HA biosynthetic pathway to achieve synchronous production of HA and HAase. Production of HA with specific molecular weights was achieved by precise regulation of the expression levels of HAase. The problem of fermentation stagnation caused by high viscosity of HA was solved by coupling the production of HA and HAase, thus greatly increasing the production efficiency. The present invention for the first time achieved efficient synthesis of HA with specific molecular weights, which has potential for bringing great economic gains in industrial applications.